WO2021007635A1

WO2021007635A1 - Process for obtaining a freeze-dried composition of chitosan-based nanoparticles containing bromelain, freeze-dried composition of chitosan-based nanoparticles containing bromelain and use in the treatment of wounds

Info

Publication number: WO2021007635A1
Application number: PCT/BR2020/050256
Authority: WO
Inventors: Janaína Artem ATAÍDE; Danilo Costa GERALDES; Laura de Oliveira NASCIMENTO; Priscila Gava MAZZOLA
Original assignee: Universidade Estadual De Campinas
Priority date: 2019-07-13
Filing date: 2020-07-13
Publication date: 2021-01-21
Also published as: BR102019014520A2

Abstract

The present invention relates to a process for obtaining a freeze-dried composition of chitosan nanoparticles containing bromelain. Said composition was seen to be stable in relation to enzyme activity using small molecules, such as sugars or amino acids, as freeze-drying additives. In addition, the present invention discloses the use thereof in preparing a medication for treating wounds.

Description

PROCESS OF OBTAINING A LYOPHILIZED COMPOSITION OF CHITOSAN-BASED NANOPARTICLES CONTAINING BROMELINE, LYOPHILIZED COMPOSITION OF CHITOSAN-BASED NANOPARTICLES CONTAINING WASTE TREATMENT

Field of the invention:

[001] The present invention relates to a process for obtaining a lyophilized composition of chitosan-based nanoparticles containing bromelain.

[002] Said composition proved to be stable in relation to the enzymatic activity using sugars as lyoprotectants.

[003] The referred composition does not contain organic solvents or chemical additives, as preservatives, being obtained by green process and based on natural and biocompatible excipients.

[004] Additionally, the present invention reveals its use in the preparation of a medicament to treat wounds.

Fundamentals of the invention:

[005] Bromelain, a complex of substances extracted mainly from pineapple (Ananas comosus L.) _r is recognized for its anti-inflammatory, antithrombotic, fibrinolytic properties, antitumor activity and immunomodulatory effect. Based on Seligman's study that showed, in 1962, its action as an anti-inflammatory agent, several studies support the use of bromelain extracts in different conditions (Seligman, 1962; Taussig and Batkin, 1988;

Salas et al. , 2008; Chobotova et al. , 2010; Amid et al. ,

2011; Ferreira et al. , 2011). In particular, some studies have demonstrated the potential use of bromelain in healing processes. Maurer (2001) demonstrated that this enzyme has benefits for wound healing, specifically reducing edema, bruising and pain. In burns, bromelain acts by hydrolyzing devitalized tissue, both in vivo and in vitro, which increases the healing capacity.

[006] Wounds are formed from the disruption of the integrity of the skin, mucous surfaces or organ tissues (Young and McNaught, 2011), and although skin lesions vary, they have a common mechanism for repair and healing. Wound healing is a regular biological process in the human body, since human skin has a natural ability to promote self-regeneration after damage (Guo and DiPietro, 2010; Pereira and Bártolo, 2016), and when triggered by a injury, this mechanism comprises an elaborate cascade of physiological events designed to finally heal the skin (Strondtbeck, 2001).

[007] The first stage, hemostasis, occurs immediately at the time of the injury and usually lasts a few hours. The second stage, inflammation, begins shortly after hemostasis and is usually completed within the first 24 to 72 hours after the injury; however, it can last up to 5-7 days after injury (Haas, 1995). Proliferation and repair, the third stage, typically occurs 1 to 3 weeks after injury. The fourth and final stage, remodeling, begins approximately 3 weeks after the injury and can take months to several years to reach physiological completion. [008] In 1962, Winter concluded that hydrated wounds in piglets epithelize the skin twice as fast as wounds exposed to air (Winter, 1962). Since then, much has been learned about wound healing mechanisms and factors that affect them (Cooper, 1990; Cuzzell and Stotts, 1990; Salas Campos et al., 2005; Winter and Scales, 1963). Currently, it is known that healing is a complex process, which can be accelerated and improved by the use of techniques, products and dressings (Cooper, 1990; Kumar et al., 2007).

[009] Modern dressings were developed to facilitate the healing process instead of just covering the wound (Dhivya et al., 2015), and its essential feature is to retain and create a moist environment around the wound to facilitate its healing (Boateng et al., 2008). Most modern treatments are interactive dressings, as they interact with the wound to provide an optimal environment at the dressing interface (Sarabahi, 2012). These interactive dressings include semipermeable film and foam, hydrofibers, hydrogels, hydrocolloids and alginates (Dhivya et al., 2015; Sarabahi, 2012).

[010] Bioactive or biological dressings are also modern dressings and are produced from biomaterials, which play an important role in the healing process (Boateng et al., 2008; Dhivya et al., 2015). These dressings are known for their biocompatibility, biodegradability and non-toxic nature (Dhivya et al.,

2015), and are generally derived from natural or artificial (Bartlett, 1981; Boateng et al., 2008; Dhivya et al., 2015). In some cases, bioactive dressings can be incorporated with active compounds, such as antimicrobials and growth factors, improving the healing process (Boateng et al., 2008; Dhivya et al., 2015).

[011] Due to the characteristics exposed above, bromelain has potential applications in the cosmetic, pharmaceutical and food industries. However, the use of proteins as an active ingredient in pharmaceutical products is a challenge, due to its physical and chemical instability.

[012] The processes to which proteins are subjected generate stress conditions that can lead to the reduction or loss of biological activity, or even alter their immunogenic potential (Sanchez-Ruiz and Makhatadze, 2001). Pereira et al. (2014) demonstrated that compositions with bromelain, when stored at 37 ° C, lost almost all of their enzymatic activity. They attributed this to the self-degradation (autolysis or self-digestion) of bromelain, as 37 ° C has been considered the optimal temperature for its proteolytic activity (Pereira et al., 2014).

[013] In recent years, the development of biodegradable nanoparticulate systems for drug delivery has grown significantly. These colloidal carriers have several advantages, such as the possibility of protecting the embedded asset against in vivo degradation, relative stability in biological fluids and the ability to modulate the release of the drug and, therefore, are considered quite promising (Lemarchand et al., 2004;

Le Droumaguet et al., 2012).

[014] Due to its characteristics of adhesiveness, biocompatibility, biodegradability and low toxicity, chitosan is an attractive material for several uses, mainly in the pharmaceutical area. This polysaccharide is used in modified drug delivery systems of various therapeutic classes, such as antibiotics, anti-inflammatories, antihypertensives, in addition to peptides and proteins (Bernkop-Schniirch, 2000; Flórea et al., 2006; Boonyo et al. ., 2007; Sandri et al., 2010). The ease of adherence of chitosan, as well as its antifungal, bacteriostatic character and its permeability to oxygen are very attractive properties for topical use (Arguelles, 2004; Jayakumar et al., 2011).

[015] It is known that the use of chitosan for the treatment of wounds and burns has been studied, based on its hemostatic capacity and its accelerating effect on wound repair with a better final aesthetic effect.

[016] Based on the above, previous studies have shown instability of bromelain when applied directly as topical compositions, even when stored at low temperatures (Lourenço et al., 2016; Pereira et al., 2014; Spir et al., 2015). Therefore, it is understood that this enzyme could benefit from chitosan-based nanostructuring for such purposes.

[017] For economic and manufacturing reasons, a new product in a liquid composition would be the simplest alternative, but possible chemical, physical and biological processes can occur in order to impair its stability and consequent performance.

[018] The lyophilization process provides solid compositions without heating above room temperature, which provides the stability of the proteins present. The solid state of low humidity prevents or decreases possible chemical, biological reactions and mechanical stress, which can result in stable compositions over the period of months to years without loss of the desired performance.

[019] The present invention therefore aimed to encapsulate bromelain in chitosan nanoparticles and lyophilize the composition in order to increase enzymatic stability, in order to produce a drug to treat wounds.

[020] Unexpectedly, it was revealed that the process comprising the use of a composition based on chitosan-bromelain nanoparticles containing 1ioprotectants, and lyophilized maintained stable particle parameters, resulting in a composition with a short time of reconstitution in water and a higher rate incorporation of bromelain when compared to the liquid form.

State of the art:

[021] Some state-of-the-art documents refer to natural assets for wound care. These assets known in the literature are propolis, Centella asiatica, Púnica granatum L, Rosmarinus officinalis L and Calendula officinalis, among others. Bromelain, whose properties also make it ideal for wound care, is extensively revised, Ataide et al. Natural actives for wound healing: a review. Phytotherapy Research, 2018.

[022] Ekambaram et al. (2017) describes a multi-phase combined distribution system produced by coaxial electrospinning of different biocompatible polymers, with optimization of the ratio and specificity of the polymers for the specific function, using bromelain as a debridement and salviolic acid B as a stimulator of angiogenesis and reepithelization. The in vitro release profile illustrated the sustained release of debridement protease and bioactive component in a timely manner. The manufactured product showed angiogenic potential through the migration of endothelial cells in vitro and an increase in new capillaries from the existing blood vessel in response to a chicken in ovo chorioallantoic membrane assay. With the projected fiber, accelerated wound healing in vivo was also achieved in the full-thickness rat skin wound model.

[023] Other documents refer to the use of bromelain associated with chitosan in nanoparticles.

[024] The article "BROMELAIN-IMMOBILIZED AND LACTOBIONIC ACID-MODIFIED CHITOSAN NANOPART _Í CLES FOR ENHANCED DRUG PENETRATION IN TUMOR TISSUES" (2018) deals with the use of a chitosan modified with lactobionic acid, which modifies its properties. The modified chitosan was then used to produce a dispersion of nanoparticles containing doxorubicin, which were post coated with bromelain _O steriormente to increase their tumor penetration ability after degradation of the tumor extracellular matrix. In this work, the composition stability was not studied. The present invention, on the other hand, addresses the problem of the stability of bromelain by proposing a composition in which the chitosan-bromelain nanoparticles are dried by lyophilization in the presence of lyoprotectant composition. In addition, the present invention does not deal with chemically modified chitosan, which reduces the excipient cost and maintains the natural appeal of the composition.

[025] CN 107475226 discloses a bromelain complex with a polyanionic polysaccharide (alginate, pectin, CMC, gum arabic or xanthan gum), and a polysaccharide or mixture may be used. According to the patent summary, the inventors complex bromelain with an anionic polysaccharide, thereby increasing its stability. However, the drying process of the complex, its average size and characteristics is not detailed. Unlike the above, the present invention makes use of a polycationic polysaccharide (chitosan has a positive charge), in addition to forming and characterizing nanoparticles with bromelain.

[026] Some state-of-the-art documents also describe obtaining lyophilized bromelain powders.

[027] CN 1834239 proposes a method for obtaining lyophilized bromelain powders. The enzyme is extracted from the pineapple in stages that involve filtering the juice fruit, adsorption with zinc oxide, ultrafiltration and lyophilization. The innovation is related to the purification method achieved by using zinc oxide, and is intended for application in possible oral compositions. However, data on stability, storage and conditions under which lyophilization is carried out are not disclosed, although these impact the enzyme activity of the product.

[028] In the present invention, there is no need to purify the extract because bromelain is commercially acquired in its purified form. In addition, the product consists of a pharmaceutical composition of bromelain encapsulated in chitosan, presented as dry powder (lyophilized), stable at room temperature for more than 90 days in relation to particle size and enzymatic activity.

[029] Document RU 2677232 refers to a pharmaceutical composition of bromelain encapsulated in low and / or high molecular weight chitosan gel, in the form of macroparticles. The bromelain is immobilized in 50 mM Tris-glycine buffer solution and pH 8, 5-9.0, incubated and the resulting precipitate is washed with 50 mM Tris-HCl buffer and pH 7.5. The invention is intended for the treatment of wounds, providing macroscopic particles of varying sizes. However, it does not present data about the physicochemical characterization of the particles or even about the stability of the composition or the lyophilization conditions.

Additionally, the composition has a final pH between 8.5 and 9.0, unsuitable for use on the skin, pH 5.5.

[030] The present invention differs in that it is a reproducible composition of lyophilized low molecular weight chitosan nanoparticles. Data on the stability of the composition, as well as the characterization of the nanoparticles, are provided and it has a pH compatible with that of the skin.

[031] It is also known that the addition of lyoprotectants, among them, sugars, are addressed in the state of the art. In particular, the article "FREEZE-DRYING OF NANOPARTYCLES: FORMULATION, PROCESS AND STORAGE CONSIDERATIONS" (2006) compiles information on the freeze-drying of nanoparticles. Maltose is mentioned for stabilizing vectors encapsulated with caprolactone, but no reference is made to its performance in terms of protein delivery.

[032] Therefore, no document describes the lyophilization of bromelain nanoparticles on the basis of chitosan with the addition of maltose as a lyoprotectant, nor does it offer extensive and detailed descriptions of the process capable of making it reproducible and increasing enzymatic stability.

Brief description of the invention:

[033] The present invention relates to a process for obtaining a lyophilized composition of chitosan-based nanoparticles containing bromelain.

[034] Said composition proved to be stable in relation to the enzymatic activity using sugars as lyoprotectants.

[035] Additionally, the present invention reveals its use in the preparation of a medicine to treat wounds.

[036] The composition obtained in the form of lyophilized powder of nanostructured bromelain can be used as a final pharmaceutical form or as an active pharmaceutical ingredient incorporated in another product.

Brief description of the figures:

[037] To obtain a complete view of the object of this invention, the figures to which reference is made are presented, as follows:

Figure 1. Scanning (A) and transmission (B) electron microscopy of chitosan-bromelain nanoparticles.

Figure 2. Infrared spectra by Fourier transform (FTIR) of bromelain (black), chitosan nanoparticles (red) and chitosan-bromelain nanoparticles (green).

Figure 3. Average size of the nanoparticles (A), polydispersion index (B) and zeta potential (C) during an accelerated stability study, in which Chi = chitosan nanoparticles, Chi-brom = chitosan-bromelain nanoparticles. Error bars represent standard deviation of three measurements.

Figure 4. Protein concentration (A) and enzyme activity (B) in the suspension of chitosan-bromelain nanoparticles during an accelerated stability study, in which Chi-brom = chitosan-bromelain nanoparticles. Error bars represent standard deviation of three measurements.

Figure 5. Bromelain encapsulation efficiency in nanoparticles according to protein concentration (A) and enzymatic activity (B), where Gly = glycine, Malt = maltose. The error bars represent the standard deviation of the three measurements.

Figure 6. Pareto graph of the factorial design relating the variables (inputs) studied in relation to the mean particle size responses (A), polydispersity index (B), Dio (C), D ₅₀ (D), D ₉₀ ( E), zeta potential (F) and encapsulation efficiency in terms of proteins (G) and enzyme activity (H).

Figure 7. Graphs of the main effect of factorial design relating the variables (inputs) studied in relation to the responses of mean particle size (A), polydispersity index (B), D ₁₀ (C), D ₅₀ (D), D ₉₀ (D), zeta potential (F) and encapsulation efficiency in terms of proteins (G) and enzyme activity (H).

Figure 8. Scanning electron microscopy of lyophilized powder with glycine (A), maltose (B) and after reconstitution with glycine (C) and maltose (D).

Figure 9. Transmission electron microscopy of lyophilized samples after reconstitution containing glycine (A) and maltose (B).

Figure 10. Average size of the nanoparticles (A), polydispersion index (B) and zeta potential (C) after reconstitution of the lyophilisate of the chitosan-bromelain nanoparticles during the stability study, where Gly = glycine, Malt = maltose. Error bars represent standard deviation of three measurements. Figure 11. Protein concentration (A) and enzyme activity (B) after reconstitution of the lyophilisate of the chitosan-bromelain nanoparticles during the stability study, where Gly = glycine, Malt = maltose. Error bars represent standard deviation of three measurements.

Figure 12. Graph of the drying ramp used during the lyophilization process of the formulation.

Detailed description of the invention:

[038] The present invention relates to the process of obtaining a lyophilized composition based on chitosan nanoparticles containing bromelain.

[039] Said composition proved to be stable in relation to the enzymatic activity using sugars as lyoprotectants.

[040] Additionally, the present invention reveals its use in the preparation of a medicament to treat wounds.

[041] The composition obtained in the form of lyophilized nanostructured bromelain powder can be used as a final pharmaceutical form or as an active pharmaceutical ingredient incorporated in another product

[042] More particularly, said process comprises steps which are described as follows:

i. Production of chitosan-bromelain nanoparticles ii. 2) Freeze-drying of chitosan-bromelain nanoparticles.

[043] Each of the steps is best described and defined below:

i. Production of chitosan-bromelain nanoparticles a) Add by dripping a solution of TPP in the concentration of 0.5 mg / mL to a solution of chitosan in the concentration of 2.5 mg / mL with stirring until complete homogenization;

b) Add a solution of bromelain at a concentration of 10mg / mL with stirring the solution obtained in step (a) until complete homogenization;

c) Obtain chitosan and bromelain nanoparticles;

ii. Lyophilization of chitosan-bromelain nanoparticles d) Solubilize the lyoprotectant directly in the solution of nanoparticles obtained in step (c);

e) The mixture of the nanoparticles with the lyoprotectant obtained in step (d) stored in an ultrafreezer at -80 ° C;

f) The nanoparticles frozen at -80 ° C, obtained in step (e) were inserted into the lyophilizer with the shelf set at -45 ° C and maintained until reaching the same temperature as the shelf;

g) The nanoparticles of step (f) follow the drying ramp, increasing the temperature by 5 ° C from -45 ° C to +10 ° C;

h) Obtaining the lyophilized composition of chitosan-based nanoparticles containing bromelain.

[044] The proportion of chitosan, TPP, bromelain being 4: 6: 1 (v / v). The agitation in steps (a) and (b) will be at 350 rpm, and the duration time in step (b) will be 40 min.

[045] In this process, the lyoprotectant is selected from small molecules, such as sugars or amino acids, maltose, glycine, preferably maltose.

[046] In step (g) the temperature is changed when the temperature of the composition reaches the temperature of the shelf, only advancing to the next step when the temperature of the formulation reaches the temperature of the shelf.

[047] The nanoparticles obtained in step (c) of chitosan-bromelain have an average diameter of 84.5 to 100.9 nm, a polydispersity index of 0.18 to 0.23, a zeta potential of 21.9 to 27 , 1 mV and a particle concentration in the order of 1012 particles / mL. Still in step c, bromelain is encapsulated in chitosan nanoparticles and has an encapsulation efficiency of 83.4 to 97.8% of the protein concentration, which corresponds to an encapsulation efficiency of 75.5 to 81.6% enzyme activity.

[048] In an embodiment of the present invention in step (d) the lyoprotectant is in a concentration of 3% (m / v) in relation to the volume of the chitosan-bromelain nanoparticle solution.

[049] In the process of the present invention the composition has a collapse temperature ranging from -56 ° C to - 28 ° C, preferably above -40 ° C, with the addition of maltose and glycine, respectively.

[050] A lyophilized composition of chitosan-based nanoparticles containing lyophilized bromelain still obtained by the process as defined above and which comprises 0.9% to 1.0% bromelain, 0.9% to 1, 0% chitosan, 3% lyoprotectant. [051] The said composition comprises a lyoprotectant selected from small molecules, which may be sugars or amino acids such as maltose or glycine, preferably maltose.

[052] Additionally, the composition object of the present invention comprises nanoparticles with an average diameter of 88.5 and 127.8 nm, a polydispersity index of 0.29 to 0.34, a zeta potential of 20.0 to 23.2 mV .

[053] The composition obtained by the process is in the form of lyophilized powder, has a maltose encapsulation efficiency of 96.3% to 98.8%, which is equivalent to an enzymatic activity of 73.3 to 99.5%.

[054] It is still an object of the present invention to use the composition obtained as defined in the process described in the preparation of a medication to treat wounds.

Example of embodiment:

1— stage: Production of chitosan-bromelain nanoparticles

Materials

[055] Pineapple stem bromelain, azocasein, Bradford reagent and low molecular weight chitosan, preferably 50,000-190,000 Da (based on viscosity) 75-85% deacetylated (catalog number 448869) were purchased from Sigma-Aldrich® (St Louis, USA). All other reagents were purchased analytically. As azocasein is the reagent used in the enzyme activity test and Bradford's reagent is the reagent used in the protein concentration test. [056] The bromelain solution was prepared by dissolving the bromelain in distilled water (10 mg / mL) and filtering it with 0.22 pm membranes.

[057] The chitosan solution was prepared in the following proportion 2.5 mg / mL of chitosan in 1% acetic acid, pH 5.0, and subsequently filtered through 0.45um membranes.

[058] The TPP solution was prepared in the following proportion 0.5 mg / mL in ultrapure water, then filtered through a 0.22 µm membrane. The TPP solution is 30% (w / w) in relation to the total amount of chitosan.

[059] The nanoparticles were produced by the ionic crosslinking technique (Shu and Zhu, 2000), using sodium tripolyphosphate (TPP) as a crosslinking agent. A proportion of 30% (w / w) of TPP was used in relation to the total amount of chitosan, and agitation of 350 rpm. The bromelain solution (10 mg / mL in water) was added under stirring at 350 rpm for 40 minutes, after dripping the TPP solution into the chitosan solution. Immediately after adding TPP, 1 ml of the bromelain solution was added to produce the bromelain-chitosan nanoparticles or 1 ml of distilled water to produce chitosan nanoparticles.

[060] Since the proportions of the solutions used in carrying out the invention, without, however, restricting this modality, were 4 ml of chitosan solution + 6 ml of TPP solution + 1 ml of bromelain solution.

[061] Nanoparticles with and without bromelain produced with different types of chitosan were physically characterized by DLS, zeta potential and NTA.

THE . Size distribution, polydispersity index and zeta potential

[062] The average diameter and polydispersity index (PDI) of the nanoparticles were evaluated using the Zetasizer Nano ZS equipment (Malvern, United Kingdom). The average size and polydispersity index were determined by dynamic light scattering (DLS), while the zeta potential was measured by Doppler laser microelectrophoresis (Zetasizer Nano ZS, Malvern, United Kingdom).

[063] All analyzes were done in triplicate of readings.

[064] Through DLS measurements, chitosan-bromelain nanoparticles showed a smaller size than chitosan nanoparticles and a reduction in zeta potential (Table 1).

Table 1. Size distribution of chitosan-bromelain nanoparticles. The results are presented as mean ± standard deviation of three measures. DLS = dynamic light scattering; NTA = nanoparticle tracking analysis; Z-ave = medium size, Dio = 10% percentile; D50 = 50% percentile; D90 = 90% percentile.

B . Encapsulation efficiency

[065] For the determination of encapsulation efficiency, the nanoparticles were ultracentrifuged (Centrifuge 5810R, Eppendorf, Germany) at 4,000g for 10 minutes, using 0.5 mL ultrafiltration devices with 100 kDa membrane (Amicon® Ultra 100k, Millipore, Germany) thus separating the bromelain that remained free from the encapsulated one. Total protein concentration and enzyme activity were determined in the initial bromelain solution and in the filtrate (resulting from the described filtration), and the efficiency was calculated according to equation 1:

Equation 1

[066] The initial bromelain solution showed 2.0 ± 0.3 mg / mL of total protein and 23.3 ± 1.6 U / mL. Protein concentration and enzyme activity were also determined in the resulting filtered solution (0.25 ± 0.01 mg / mL and 4.5 ± 0.6 U / mL, respectively) to calculate the encapsulation efficiency, which was 87.0 ± 5.1% of the total protein, corresponding to 80.7 ± 1.1% of the enzymatic activity of bromelain (Table 2).

Table 2. Total protein concentration, enzyme activity and bromelain encapsulation efficiency

C. Scanning and transmission electron microscopy

[067] The morphological characteristics of chitosan-bromelain nanoparticles were observed using a Leo 440i scanning electron microscope with a 6070 X-ray dispersive energy detector (LEO Electron Microscopy, England). Scanning electron microscopy images were obtained using an acceleration voltage of 15 kV. Before analysis, the samples were dried at room temperature, under vacuum, for 24 hours and coated with gold (92 A °) using SC7620 Sputter Coater Polaron (VG Microtech, England). The particles were also observed in a transmission electron microscope (TEM JEOL-1400 Plus, 120kV), and the grids were prepared by negative contrast.

[068] On scanning electron microscopy image (Figure IA), the chitosan-bromelain nanoparticles had a spherical shape, smooth surface and diameter of approximately 1 pm, which was larger than the size obtained by the DLS and NTA measurements. Showing that even a gentle drying process, at room temperature, can lead to the aggregation of nanoparticles, leading to the formation of microparticles (Rampino et al., 2013). In the transmission electron microscopy image (Figure 1), the particles continue to have a spherical shape with a diameter of less than 50nm, however it is possible to notice the presence of clusters with several particles joined with approximately 100nm, which is consistent with the results obtained by DLS and NTA.

D. Infrared spectroscopy with Fourier transform

[069] Infrared spectra of chitosan and chitosan-bromelain nanoparticles, and free bromelain (Figure 2), were obtained from an infrared spectrophotometer with Fourier transform (Shimadzu Scientific Instruments, Model 8300, Japan), operating from 4000 at 650 crrr ¹ , with a resolution of 4 cm ^-1 . All measurements were performed in triplicate.

[070] In the spectrum of bromelain (black) a peak at 3280 cm-1 can be observed that corresponds to the enzymatic peptide bond. It is also possible to observe a peak at 1634 cm-1 indicative of group C = 0, and at 1515 crrr ¹ indicative of NH group, which confirm the presence of amino acids. The peak in 1236 cm-1 can be attributed to the CN bond, in aliphatic amine (Devakate et al., 2009; Soares et al., 2012; Ataide et al., 2017a; Ataide et al., 2017b).

[071] The spectra of the nanoparticles show an absorption band around 3400 crrr ¹ , equivalent to the interaction of hydrogen and OH vibration (Wu et al., 2005), with greater intensity in the chitosan-bromelain nanoparticle when compared to the chitosan nanoparticle . In both spectra there is also a peak around 795 crrr ¹ , which can be attributed to the presence of vibrations related to the connection between phosphorus and oxygen (P-0 and POP) (Knaul et al., 1999;

Antoniou et al. , 2015). In the chitosan nanoparticle

(red) it is possible to observe the displacement of the peaks of vibration C = 0 of amide II and NH of amine I, which indicates the interaction between the amino groups of chitosan with the phosphate groups of TPP (Knaul et al., 1999; Antoniou et al., 2015). Comparing this same region of the chitosan nanoparticle and the chitosan-bromelain nanoparticle (green), there is an increase in intensity of both peaks, which can be attributed to the presence of bromelain, since bromelain also absorbs at the same frequency (Devakate et al., 2009;

Soares et al. , 2012). In addition, again, the displacement of the peaks corresponding to the N-H group is observed, which indicates interaction with the amino group.

E. Stability studies

[072] For the stability study of nanoparticles, 10 mg / mL bromelain solution and chitosan and chitosan-bromelain nanoparticles. The 3 solutions were conditioned in glass bottles and stored in 4 conditions: light environment (27 ± 2 ° C, with exposure to light), dark environment (27 ± 2 ° C, protected from light), refrigerator (5 ± 2 ° C) and greenhouse (45 ± 2 ° C). For the preliminary stability test, the samples were evaluated for 15 consecutive days, starting on day 0 (which were prepared and packaged). The accelerated stability test was performed in sequence, under the same conditions, evaluating the samples on days 30, 45, 60, 90, 120, 150 and 180 (Anvisa, 2005).

[073] During the preliminary stability study, general parameters, such as pH value and macroscopic characteristics were evaluated. The suspension of chitosan and chitosan-bromelain nanoparticles did not show macroscopic and pH changes. Thus, the samples continued in the accelerated stability study. After 7 days of study (Figure 3), the chitosan-bromelain nanoparticles showed a significant increase (p <0.05, Student's T test) in the average diameter in all conditions studied, also accompanied by a significant increase in the polydispersion index and decreased zeta potential. The increase in particle size is regularly attributed to particle agglomeration, which can be induced by the adsorption of active molecules in the nanoparticles (Magenheim and Benita, 1991; Abdelwahed et al., 2006).

[074] Protein concentration and enzyme activity were also determined during the stability study accelerated. Chitosan nanoparticles did not show significant protein concentration or enzyme activity in any time or condition studied. Chitosan-bromelain nanoparticles showed a decrease in protein concentration and enzyme activity (Figure 4) over time, where enzyme activity decreased significantly (p <0.05, Student's T test) after 7 days. This decrease was slightly less pronounced when the samples were stored at low temperatures (Figure 4), according to results reported by other studies that used bromelain (Pereira et al., 2014; Spir et al., 2015; Lourenço et al. , 2016). Given these results, chitosan-bromelain nanoparticles are unstable when stored in liquid form, and therefore lyophilization is an important process to increase the preservation of particle size for long-term storage (Fonte et al., 2016; Almalik et al., 2017) and the enzymatic activity of bromelain (Arakawa et al., 2001).

Step 2: Freeze-drying of the nanoparticles of bromelain quitosana-

THE . Assessment of lyoprotectant addition and collapse temperature

[075] Trealose, maltose and glycine were chosen as possible lyoprotectants, due to their collapse temperatures when alone in solution, with the collapse temperature being -24.3 °, -25.1 and -12.7 ° , respectively. Subsequently, they were dissolved in the suspension of chitosan-bromelain nanoparticles at a concentration of 3% (m / v), to evaluate their effect on the collapse temperature of the suspensions of chitosan-bromelain nanoparticles. The collapse temperature of chitosan-bromelain nanoparticles with and without lyoprotectants was determined by a microscope attached to a lyophilization module, Lyostat 2, model FDCS 196 (Linkam Instruments, Surrey, United Kingdom), equipped with a liquid nitrogen freezing system (LNP94 / 2) and programmable temperature controller (TMS94, Linkam). The chitosan-bromelain nanoparticles showed a collapse temperature of -56 ° C, which decreased to -49.3 ° C, -34.8 ° C and -28.0 ° C with the addition of trehalose, maltose and glycine, respectively .

[076] Although the two sugars (maltose and trehalose) have close collapse temperatures when alone (Table 3), it was unexpected that they would result in solutions with such different collapse temperatures when in conjunction with the dispersion of nanoparticles.

Table 3 Collapse temperature of possible lyoprotectants in isolated solution and in nanoparticles.

[077] In general, the freeze-drying process below

40 ° C is not advisable (Carpenter et al., 1997; Tattini Jr et al., 2006) and, thus, maltose and glycine were evaluated in factorial design as lyoprotectants, since they allow lyophilization to begin - 40 ° C.

B . Experimental planning of the lyophilization process

[078] To optimize the lyophilized composition, of the experimental points, only with the nanoparticles a factorial design 2 ³ was carried out with chitosan-bromelain duplicate. The variables (inputs) evaluated were the type of lyoprotectant (glycine or maltose), the lyoprotectant concentration (3 or 5%, m / v) and the lyophilization process itself (before and after the process). The samples were subjected to rapid freezing with liquid nitrogen and lyophilized in Lyostar 3 Freeze-Drier (SP Scientific, USA). Primary drying was carried out by increasing the temperature from -45 ° C to 10 ° C by 5 ° C (Figure 12), waiting for the temperature of the vials to reach the temperature of the shelves. After drying, the samples were reconstituted with distilled water for the same initial volume and were physicochemically characterized by dynamic light scattering and encapsulation efficiency, which were used as factorial design responses. The lyophilized products were subjected to thermogravimetric analysis

(TGA-50M, Shimadzu, Japan) in order to determine residual moisture. The samples of dried nanoparticles were precisely weighed (about 10 mg) and were then heated at 10 ° C / min from 25 ° C to 300 ° C, under a nitrogen atmosphere with a flow rate of 50 ml / min. Residual humidity was determined by stable weight loss (%) at a temperature of about 100 ° C (Sylvester et al., 2018).

[079] All lyophilisates looked good and did not shrink. The nanoparticles with glycine and maltose were immediately reconstituted after adding water and the resulting solutions were clear. Without the lyoprotectant, however, resuspension took more than 20 seconds and the solutions were slightly cloudy, even after vortexing. Lyophilized powders were analyzed by thermogravimetric analysis to determine residual moisture. Compositions with glycine in concentration of 3% and 5% showed 4.1% and 3.2% of loss of mass, respectively, while the compositions with maltose presented 5.6% and 3.8%. The lyophilizates were reconstituted in distilled water to analyze the physical characteristics of the particles by dynamic light scattering (Table 4) and the encapsulation efficiency (Figure 5), which were considered outputs for factorial design. After lyophilization, glycine in a concentration of 3% (m / v) was able to keep the polydispersity index within the desirable values, however the zeta potential declined. On the other hand, maltose showed the opposite behavior, increasing the polydispersity index and showing little effect on the zeta potential. Table 4. Size of nanoparticles, PDI and zeta potential before and after the lyophilization process (experimental design). The results are presented as mean ± standard deviation of two compositions, measured three times each. Z-ave = mean particle size, PDI = polydispersity index, control = nanoparticle without lioprotector.

[080] The encapsulation efficiency before the lyophilization process increased in terms of protein concentration after the addition of glycine and maltose, while the encapsulation efficiency in terms of enzymatic activity decreased after the addition of glycine and increased after the addition of maltose (Figure 5). After lyophilization, it was possible to observe a small increase in the protein encapsulation efficiency for all samples. The encapsulation efficiency in terms of enzyme activity has also increased in all compositions. [081] The responses (outputs) (mean particle size, PDI, D10, D50, D90, zeta potential and encapsulation efficiency) were statistically analyzed using pareto diagrams (Figure 6), and the main effects of variables on responses were also analyzed (Figure 7). Lyoprotectants significantly interfere with almost all responses, excluding zeta potential and encapsulation efficiency in terms of enzyme activity. Glycine increased the average particle size by increasing the D10, D50, D90 distribution percentages, however it also decreased the polydispersity index, indicating a narrower size distribution. On the other hand, maltose decreased the average size of the nanoparticles and increased the polydispersity index. The concentration of lyoprotectants was an important variable observed for PDI, D10 and protein encapsulation, with low concentrations (3%, m / v) decreasing PDI, increasing D10 and protein encapsulation.

[082] Excluding protein encapsulation, lyophilization significantly affects all responses. The process increased the average particle size by increasing the percentile of distribution, also increasing the polydispersity and the encapsulation of enzymatic activity. The zeta potential decreased with the lyophilization process, which could cause some problem in the stability of the nanoparticles, which must be studied. Considering all the effects, the compositions with glycine or maltose the concentration 3% (w / v) seem to have the potential to better stabilize chitosan-bromelain nanoparticles.

C. Production and characterization of lyophilized nanoparticles

[083] Based on factorial design, it was found that compositions containing 3% (w / v) of glycine and maltose as lyoprotectants were the most promising for stabilizing chitosan-bromelain nanoparticles. Thus, a new batch of samples with these excipients was produced and lyophilized following the parameters previously determined. Immediately after the end of the lyophilization process, the samples were characterized and submitted to a stability study. For this study, the dried samples were kept sealed in the lyophilization vials with plastic wrap and stored at room temperature (25 ° ± 2 ° ) and refrigerator (5 ± 2 ^o) protected from light.

[084] The samples were characterized in terms of average size, polydispersity index and zeta potential, before and immediately after lyophilization (Table 5). The addition of glycine increased the average size of the nanoparticles and the zeta potential and decreased the polydispersity index when compared to the nanoparticles without the addition of lyoprotectants. On the other hand, the addition of maltose has the opposite effect, decreasing the average size and zeta potential and increasing the polydispersity index. As observed in the experimental design, after the lyophilization process the average size of the nanoparticles and the PDI increased, while the zeta potential decreased. The encapsulation efficiency in all cases increased after the lyophilization process.

Table 5. Size of nanoparticles, PDI and zeta potential before and after the lyophilization process. The results are presented as mean ± standard deviation of two compositions, measured three times each. Z-ave = mean particle size, PDI = polydispersity index, EE Ptn = total protein encapsulation efficiency, EE Activ encapsulation efficiency enzymatic activity.

[085] After the lyophilization process was finished, the morphology of the samples was evaluated by scanning electron microscopy (Figure 8) in dry form (Figure 8A and * B) and after reconstitution (Figure 8C and 8D). Due to equipment limitation, samples after reconstitution have small dots, and it is not possible to determine the nanoparticles limit properly.

[086] The reconstituted samples were also analyzed using a transmission electron microscope (Figure 9). In these images it is possible to notice that both particles have a spherical shape, and the particles lyophilized with glycine have a larger average diameter and a more homogeneous size distribution when compared to the composition containing maltose. However, the maltose composition preserved more the characteristics of the initial suspension of chitosan-bromelain nanoparticles, that is, the presence of agglomerates of approximately 100nm composed of several smaller particles.

[087] The samples showed residual moisture of 3.5% and 4.4% for the samples with glycine and maltose respectively, which were lower than the values found during planning, but still above the recommended limit of 2% (Abdelwahed et al., 2006; Sylvester et al., 2018). After 90 days of stability study, the residual humidity of the samples with glycine increased to 6.2% and 7.6% when stored at room temperature and in a refrigerator, respectively. The maltose samples showed a reduction in residual moisture, which was 2.4% and 1.8% when stored at room temperature and refrigerator, respectively.

[088] On days 0, 30, 60 and 90, samples stored at room temperature and in a refrigerator were reconstituted with the same initial volume (3mL of MilliQ water) and were monitored for mean size, polydispersity index and zeta potential ( Figure 10), and protein concentration and enzyme activity (Figure 11). Despite presenting the highest polydispersity index at the beginning of the stability study, the composition of nanoparticles of chitosan-bromelain containing maltose kept the particle parameters stable throughout the study period (Figure 10), with the composition stored in a refrigerator (5 ^o ± 2 ^o ) showing the most stable results. In turn, the compositions were not stable with glycine, reaching a PDI of 1.0 when stored at room temperature (25 ° ± 2 ° ).

[089] As for the parameters related to the stability of bromelain (enzyme activity and total proteins), it was possible to notice that the protein concentration and enzyme activity did not show significant changes at the beginning and at the end of the stability study (days 1 and 90) Figure 11.

Claims

1. Process of obtaining a lyophilized composition of chitosan-based nanoparticles containing bromelain, characterized by the fact that it comprises the following steps:

i. Production of chitosan-bromelain nanoparticles

ii. Lyophilization of chitosan-bromelain nanoparticles.

2. Process according to claim 1, characterized by the fact that it comprises the following steps and substeps: i. Production of chitosan-bromelain nanoparticles

a) Add by dripping a solution of TPP in the concentration of 0.5 mg / mL to a solution of chitosan in the concentration of 2.5 mg / mL with stirring until complete homogenization;

c) Obtain chitosan and bromelain nanoparticles;

h) Obtaining the lyophilized composition of nanoparticles based on chitosan containing bromelain.

Process according to claims 1 and 2, characterized in that the proportion of chitosan, TPP, bromelain is 4: 6: 1 (v / v).

Process according to claims 1 and 2, characterized by the agitation in steps (a) and (b) being at 350 rpm.

Process according to claims 1 and 4, characterized in that step (b) has a duration time of 40 min.

6. Process according to claims 1 and 2, characterized in that the lyoprotectant is selected from small molecules, such as sugars or amino acids, maltose, glycine, preferably maltose.

Process according to claims 1 and 2, characterized in that in step (g) the temperature changes when the temperature of the composition reaches the temperature of the shelf.

8. Process according to claims 1 and 2, characterized by the fact that in step (c) the chitosan-bromelain nanoparticles have an average diameter of 84.5 to 100.9 nm, a polydispersity index of 0.18 to 0.23, a zeta potential of 21.9 to 27.1 mV and a concentration of particles in the order of 10 ¹² particles / mL.

9. Process, according to claims 1 and 2, characterized by the fact that additionally in step c, the bromelain is encapsulated in chitosan nanoparticles and has an encapsulation efficiency of 83.4 to 97.8% of the protein concentration, which corresponds to an encapsulation efficiency of 75.5 to 81.6% of the enzymatic activity.

10. Process, according to claims 1 and 2, characterized by the fact that in step (d) the lyoprotectant is in a concentration of 3% (m / v) in relation to the volume of the chitosan-bromelain nanoparticle solution.

11. Process according to claim 10, characterized by the fact that it has a collapse temperature ranging from -56 ° C to -28 ° C, preferably above -40 ° C, with the addition of maltose and glycine, respectively .

12. Lyophilized composition of chitosan-based nanoparticles containing lyophilized bromelain obtained by the process as defined in claims 1 to 11, characterized by comprising 0.9% to 1.0% bromelain, 0.9% to 1.0% of chitosan, 3% lyoprotectant.

13. Composition according to claim 12, characterized in that the lyoprotectant is selected from small molecules, which may be sugars or amino acids such as maltose or glycine, preferably maltose.

14. Composition according to claim 12 characterized by the fact that it comprises nanoparticles with an average diameter of 88.5 and 127.8 nm, a polydispersity index of 0.29 to 0.34, a zeta potential of 20.0 to 23 , 2 mV.

15. Composition according to claim 12 characterized by the fact that it is in the form of lyophilized powder.

16. Composition according to claim 12, characterized by the fact that it presents a maltose encapsulation efficiency of 96.3% to 98.8%, which is equivalent to an enzymatic activity of 73.3 to 99.5%.

17. Use of the composition obtained as defined in claims 1 to 11, characterized in that it is in the preparation of a medicine to treat wounds.

18. Use of the composition obtained as defined in claims 12 to 16, characterized in that it is in the preparation of a medicine to treat wounds.